HISTOPATH Flashcards
Dolor means
Pain
Site of collection for hormonal studies
Upper quadrant of vaginal wall
Prosector of autopsy
Pathologist
Cells with fried egg appearance
Parabasal cells
Stain for copper pigments (recall)
Lindquist rhodamine
Ratio of 10% formalin
1:9 (1 parts of stocksolution: 9 parts of water)
Most common metallic fixative
Mercuric chloride
Most rapid fixative
Carnoy’s
Pathology means
Study of suffering/pain
Tissues with intercalated disk
Cardiac muscle
Flat, polyhedral cells with a pyknotic nucleus
Superficial cells
Removes nicks
Honing
Scott’s tap water is employed for
Blueing Agent
Alkaline Fast green is useful as a stain for
Histones
Fastest method of paraffin impregnation
Vacuum
Ideal fixative to tissue ratio
20:1
-Itis means
Inflammation
Ab class used in IHC
IgG
Both a nuclear and histochemical fixative
Newcomer’s
Stain for glomerular basement membrane
PAS (Periodic Acid Schiff)
Inventor of Sliding microtome
Adams
Epon is employed as
Plastic embedding medium for Electron microscopy
Non-appearance of organ
Agenesia
Clearing agent that becomes milky
Xylene
Routine fixative for exfoliative cytology
95% ethanol
Recommended fixative for cell detail in tissue photography
Mercuric chloride
Negri bodies are seen in which viral disease
Rabies
Presence of mononuclear cells are associated with which type of inflammation
Chronic inflammation
Most reliable feature of malignancy
Metastasis
Double embedding technique
1st: Celloidin ; 2nd: Paraffin
Tissue examination can be performed in _____ or ______
Fresh or Preserved tissues
Examined in its living state;
Allows examination of protoplasmic activities;
Fresh tissues
What are the protoplasmic activities of fresh tissues?
Phagocytosis
Pinocytosis
Mitosis
Motility
(PPMM)
Tissues are not permanent:
Fresh or Preserved ?
Fresh tissues
4 Methods of FRESH TISSUES examination
Teasing/ Dissociation
Squash Preparation/Crushing
Smear Preparation
Frozen Section
Tissues is immersed in a watch glass with NSS;
Dissected or separated using needle
Teasing/ Dissociation
In Teasing, tissues is immersed in a watch glass with _____
NSS
Microscope needed in Teasing method
Phase contrast/ Bright field
Stain used in Teasing method
Methylene Blue
Small tissues are placed in a slide;
Tissues are compressed with another slide or coverslip
Squash Preparation/Crushing
In squash method, what is the size of tissue needed to be place in a slide
1mm or less
Stain used in squash method
Supravital stain
Cellular materials are spread lightly over a slide
Smear preparation
Method of fresh tissue examination:
Useful for cytology
Smear Preparation
4 types of Smear Preparation
Streaking
Spreading
Pull- Apart
Touch Preparation
Material is added using an applicator stick of loop in a direct or zigzag fashion
Streaking
Material is placed on a clean slide and spread into a moderately thick film by teasing with an applicator stick
Spreading
Advantage of spreading smear
Maintains intercellular relationship
Use of 2 slides in preparing the smear
Pull-Apart
Smear used for thick secretions
Pull-Apart
Touch preparation is A.K.A
Impression smear
Freshly cut piece of tissue in brought into contact and pressed on to the surface of a clean slide
Touch Preparation
Touch preparation is used for
Lymph nodes
The slide of touch preparation should be ____ and _____
sterile and with polished edges
For rapid diagnosis of fresh tissue examination
Frozen section
Time needed for:
Rush Frozen Section
5-15 mins
Apparatus used in Frozen section
Cryostat/Cold microtome
Freezing Microtome
Method of tissue examination:
Used for demonstration of lipids, nervous tissue elements ,enzymes
Frozen section
Preserved tissue processing:
Permanent or Not Permanent?
Permanent
Frozen section is done:
Intraoperatively or Preoperative or Postoperative
Intraoperatively
First and most important step in histopathology
Numbering (Accessioning)
In numbering, the specimen number is preceded by either:
S=
A=
C=
Surgical
Autopsy
Cytology
Example:
S99-0345, what does it mean?
345th Surgical specimen in the year 1999
The MT will then write down the description for processing at the ________
back of the request or in-front of the request ?
back of the request
Use ___ in writing the description of the tissue specimen
Pencil
Specimen size for processing:
2 x 3 cm
3-5 mm thick
What can be added for small tissues
1% eosin
For electron microscopy, specimen size should be?
1 mm^3
Most crucial step in histopath
Fixation
Process of preserving cells and tissue constituents in a condition identical to that existing during life
Fixation
Fixation prevent ____
Autolysis
Self-destruction of tissues
Autolysis
3 Methods of Fixation
Heat fixation
Perfusion
Immersion
Fixation via blood flow
Perfusion
2 Mechanism of action in fixation
Additive
Non-additive
The fixative becomes part of the tissue by formation of cross links/complexes
Additive
Stabilizes the tissue proteins
Additve
Ex: of additive fixation
Mercury
Formalin
Osmium tetroxide
Fixative not incorporated into the tissues
Non-additive
Alteration of tissue composition
Additive or Non-additive?
Non-Additive
Stabilizes the tissue by removing water
Additive or Non-additive?
Non-Additive
Ex of Non-additive fixation
Alcoholic fixative
Ex of mordant/accentuators in fixation
Osmium tetroxide
Potassium dichromate
pH of fixation
6-8
Traditional temp for fixation
Room temp
Autotech temp for fixation
40Degree Celsius
EM and Histochem temp for fixation
0-4 Degree Celsius
Rapid fixation temp
60 Degree Celsius
Temp of fixation for tissues with TB
100 Degree Celsius
Thickness of fixation for EM
1mm ^3 or
1-2 mm^2
Thickness of fixation for LM
2x3 cm or
2cm^2
Tissues should not be more than ___ thick
4-5 mm
Thickness of edematous lung tissues
10-20 mm or
1-2cm
Suspended in whole and tie with string
Brain tissues
Brain tissues should be tie at the string of ___ (recall)
Circle of Willis
Fixative for brain tissues
10% buffered formalin
Brain tissues should be fixed for how many?
2-3 weeks
Ex of large solid tissues
Uterus
Large solid tissues should be ____ to allow the fixative to enter the tissue
Cut open
Osmolality of fixation
Slightly hypertonic solution
aroung 400-450 mOsm
Ideal for immunolectron microscopy
0.25% glutaraldehyde
Time duration of primary fixation in buffered formalin
2-6 hours
Time duration of EM fixation
3 hours then placed in a holding buffer
Speed of fixation
<1hr or immediately
Why does fixation need to be done immediately
To prevent autolysis and putrefaction
Rate of penetration of formalin
1mm/hr
Volume or ratio of ideal fixation
10-20x of that tissue or 20:1 (fixative:tissue)
Volume for expensive fixative
5-10x reduced
Example of expensive fixative
Osmium tetroxide
Volume for museum preparation
atleast 50x of that tissue
Duration of tissue depends on
tissue structure
Duration of fixation of fibrous tissue
longer fixation time
Duration of fixation of small or loosely textured tissues
shorter fixation time
Duration of fixation can be hastened by:
Heat
Agitation
Vaccum
Microwave
“HAV Mi”
If autopsy materials are not anle to be fixed, these should be placed in:
Mortuary refrigeration at 4 Degree Celsius
Undergo Arterial Embalming
Organs that need to be grossed after fixation
Brains and eyes
Example of hollow organs
Stomach and intestine
Organs that tend to float in fixation
Hollow organs and Air filled lungs
Remedy for hollow organs in fixation that tend to float
Put cotton inside
Remedy for air filled lungs that tend to float in fixation
Wrap in gauze
Lungs of pneumonia patient tend to float or sink during fixation?
Sink
Due to combination of increased fluid content and consolidation in the lungs of pneumonia patients leads to a loss of buoyancy, causing them to sink during fixation.
What considereation should be done in Eyes specimen during fixation ?
Inject with formol alcohol
Example of hard tissues
Cervix,fibroids,hyperkeratotic skin, nails,teeth,bone
Consideration need to be done in hard tissues
Immersed the tissue in a softeners such as Perenyi’s or Lendrum’s before sectioning
Fixative types according to composition
Simple
Compound
Type of fixative that contains 1 fixative
Simple fixative
Type of fixative that contains 2 or more fixative
Compound fixative
Ex. of simple fixative
Heat
Aldehyde
Metallic Fixative
“HAM”
Fixatives according to action
Microanatomical
Cytological
Fixatives that permits general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues
Microanatomical
Fixative that preserve a specific part/ element of cells
Cytological
3 Types of cytological fixative
Nuclear
Cytoplasmic
Histochemical
Fixative that preserve nucleus and chromatin material and contains glacial acetic acid
Nuclear fixative
Purpose of glacial acetic acid
Increase affinity to nuclues
pH of nuclear fixative
4.6 or less
Fixative that preserves cytoplasm and organelles and no glacial acetic acid
Cytoplasmic
pH of cytoplasmic fixative
4.6 or more
Fixative that preserve chemical components
Histochemical
Example of Microanatomical fixative
Bouin’s
Brasil’s
10% Formol Saline
10% Neutral Buffered Formalin
Formol sublimate
Zenker’s
Heidenhain’s Susa
“BB FNF Z Heiden”
Example of Nuclear Fixative
Flemming’s
Carnoy’s
Bouin’s
Newcomer’s
Heidenhain’s Susa
“FC Ba Ni Heiden”
Example of Cytoplasmic Fixative
Flemmings without HOAC(Acetic acid)
Formalin with post chroming
Orth’s
Regaud’s
Kelly’s
“FFORK”
Example of Histochemical fixative
Absolute ethanol
10% Formol Saline
Acetone
Newcomer’s
“A FAN”
10% formol saline is what type of fixative
Microanatomical and histochemical
Heidenhain’s Susa is what type of fixative
Microanatomical and Nuclear
Bouin’s is what type of fixative
Microanatomical and Nuclear
Newcomer’s is what type of fixative
Nuclear and histochemical
Example of aldehyde fixative
fixative with suffix -aldehyde
And gendre’s/alcoholic formalin/alcoholic bouin
Gas produced from oxidation of methanol
Formalin/Formaldehyde
% of stock/concentrated formalin
37-40%
Routine formalin
10% formalin
Actual concentration of 10% formalin in 37-40% stock solution
4%
Formalin is usually buffered with
PO4 buffer to pH 7 - to prevent formalin pigments
How to retards decomposition to formic acid
add 10% methanol
Formalin added with 10% methanol is no longer suitable with ____
Electron microscopy
Usual fixation time of formalin
12-24 hours
The advantage of this fixative is easy to prepare,cheap,compative with many stains
Formalin
The advantage of this fixative is irritating to nose and eyes and can cause allergic dermatitis
Formalin
Use what type of gloves in preparing formalin to avoid allergic dermatitis
rubber gloves
Prolonged storage of formalin can lead to what formation
White precipitate (Paraformaldehyde)
What is the remedy for white precipitate formation in formalin?
Filtration
Add 10% methanol to dissolve
What color of precipitate form in blood contaning tissue when formalin is used
Brown or black crystalline precipitate
Brown or black crystalline precipitate is AKA
Acid Formaldehyde Hematein
Example of blood containing tissues that cause brown or black precipitate
Spleen
What is the reason of formation of brown/black precipitate
Due to the action of formic acid with blood
What is the remedy for removal of formalin pigments
1) Kardasewitsch’s method (70% ethanol & 28% ammonium water)
2) Lilie’s method (hydrogen peroxide and 28% ammonium water)
3) Picric acid method (saturated alcoholic picric acid)
4) 1% KOH in 80% alcohol
10% formol saline is diluted with
NaCl
Fixation of CNS tissues and General Post Mortem Tissues for Histochemical examination
10% formol saline
Ideal for silver impregnation
10% formol saline
Best fixative for Iron containing pigments and elatic fibers
10% neutral buffered formalin /PO4 buffered formalin
pH of PO4 buffered formalin
7
Recommended routine fixative but longer to prepare
10% Neutral Buffered Formalin / PO4 Buffered Formalin
Aldehyde fixative that contains Mercuric chloride
Formal Corrosive/ Formol Saline
Excellent for stains like Silver Reticulin Method
Formal Corrosive/ Formol Saline
Aldehyde fixative with no washing out and fixes lipids
Formal Corrosive/ Formol Saline
Has 95% ethyl alcohol with picric acid and glacial acetic acid
Gendre’s / Alcoholic formalin/ Alcoholic Bouin
AKA as Alcoholic Bouin
Gendre’s
Good for glycogen and microincineration technique
Gendre’s
Fixes Sputum
Gendre’s
Contains 2 formalin residues linked by 3 carbon chains and acts similarly to formalin
Glutaraldehyde
% of Glutaraldehyde in small and large tissues
2.5% = small tissues
4% = Large tissues
Recommended for enzyme histochem and fixative for EM
Glutaraldehyde
Primary Fixative for TEM (Tranmission Electron Microscopy)
Glutaraldehyde
Better preservation of cellular and fluid proteins
Less irritating but expensive
Glutaraldehyde
Glutaraldehyde specimen vial should be ___
refrigerated
Polymer formalin in white powrder forn
Paraformaldehyde
Concentration of Paraformaldehyde
4%
Use for thin and ultrathin sections for plastic embedding and EM
Paraformaldehyde
3 Types of Metallic Fixative
Mercuric chrloride
Chromate
Lead
Most common metallic fixative
Mercuric fixative
Concentration of metallic fixative
5-7%
What type of fixative according to component is Mercuric chloride ?
Simple or Compound
Compound fixative
Fixative that produce black granular deposit
Mercuric chloride
All mercuric chloride produce black granular deposit except
Heidenhain’s susa
Removal of black granular deposit can be done by
Dezenkerization (Washing out)
Dezekerization solution contains
Saturated iodine solution of 96% alcohol &
5% sodium thiosulfate
Metallic fixatives that penetrate and harden tissue rapidly
Mercuric chloride
Routine fixative of choice for preservation of cell detail in tissue photography
Mercuric chloride
Fixation of hematophoetic and reticuloendothelial tissues
Mercuric chloride
Example of Mercuric chloride
Zenker’s
Zenker’s formol/ Helly’s/Kelly’s
Heidenhain Susa
B5 Fixative
“ZZ H5”
Mercuric chloride that contains glacial acetic acid
Zenker’s
Recommended for small pieces of liver,spleen,CT fibers and nuclei
Zenker’s
Recommended for trichrome staining
Zenker’s
Contains Potassium Chromate and formalin
Zenker’s formol/Helly’s/Kelly’s
Excellent for microanatomical fixative of pituitary gland,BM and blood containing organ
Zenker’s formol/Helly’s/Kelly’s
Preserves cytoplasmic granules and produce brown pigments
Zenker’s formol/Helly’s/Kelly’s
what is the remedy of brown pigments due to kelly’s
Remove using picric acid or NaOH(sodium hydroxide)
It composed of TCA,Glacial cetic acid and formalin
Heidenhain Susa
Recommended for skin tumor biopsies
Heidenhain Susa
Has anhydrous Na acetate
B5 Fixative
For bone marrow biopsies and lymph nodes
B5 Fixative
3 Example of Chromate Fixatives
Potassium Dichromate
Regaud’s /Muller’s
Orth’s Fluid
“PRO”
Concentration of chromate fixative
1-2% aqueous solution
It is a strong oxidizing agents and should not be combined with reducing agents
Chromate fixative
It preserved carbohydrates
Chromate fixative
It contains 3% aqueous solution
Potassium Dichromate
Preserves lipids and mitochondria at pH 4.5-5.2 cytoplasm,chromatin and chromosomes are fixed
Potassium Dichromate
Regaud’s is AKA
Muller’s
Fixed chromatin,mitochondria,mitotic figures ,golgi bodies,rbc, and colloid containing tissues
Regaud’s
For early degenerative process and tissue necrosis
Orth’s fluid
Demonstration of Rickettsia
Orth’s fluid
Preserves myelin
Orth’s fluid
Contains 4% aqueous solution and recommended for Acid mucopolysaccharide
Lead Fixatives
Fixes mucin also
Lead Fixatives
Usually used in a strong or saturated solutions
Picric acid
Chemically known as 2,4,6 Trinitophenol
Picric acid
Can be used as stain (yellow) and small tissue fragments can be seen
Picric acid
Explosive when dry
Picric acid
Remedy for yellow stain cause by picric acid
Saturated solution of Lithium Carbonate
70% ethanol
5% Sodium Thiosulfate
Excellent for glycogen
Picric acid
Example of Picric Acid
Bouin’s
Brasil’s
Hollande’s solution
“BB Hollande”
Recommended for embryonic and pituitary biopsies
Bouin’s
Bouin’s are stained using
Masson’s trichrome stain
Not for kidney fixation
Bouin’s
Has TCA ,better and less messy than Bouin’s
Brasil’s Alcohic Picroformol fixative
For GI tract samples and endocrine tissues
Hollande’s solution
Less lysis than Bouin’s
Hollande’s solution
Decalcifying property
Hollande’s solution
Glacial acetic acid is what type of fixative?
Simple or Compound?
Compound
Glacial acetic acid solidies at
17 Degree Celsius
Not for cytoplasmic fixation
Glacial acetic acid
What will happen if Glacial acetic acid is used for cytoplasmic fixation
Cell swelling
Example of Alcoholic Fixatives
Newcomer’s
Methanol
Isopropyl
Carnoy’s
Ethanol
“New MICE”
Rapidly denatures and precipitates protein
Alcoholic fixatives
Alcoholic fixatives acts as both
Fixative and Dehydrating agent
Use for glycogen fixation
Gendre’s
Picric
Alcohol fixative
Alcohol fixatives preserves:
Nuclear stain or cytoplasmic stain?
Nuclear stain
Alcoholic fixatives dissolves
Fats and Lipids
Methanol is AKA
Wood Alcohol
Concentration of Wood Alcohol
100%
Alcohol fixatives used for blood smear, bone marrow smear, wet and dry smear
Methanol
Isopropyl Alcohol is AKA
Rubbing Alcohol
Concentration of Rubbing Alcohol
95%
Alcohol fixatives that fixes touch preparation and for wright giemsa
Isopropyl Alcohol
Ethanol is AKA
Grain Alcohol
Concentration of Grain alcohol
70-100%
All alcohol fixative is excellent for glycogen fixation except
Ethanol
Fixative useful for PCR
Ethanol
Most Rapid fixative
Carnoy’s fixative
Fixation time of Carnoy’s fixative
1-3 hours
Fixative that contains Absolute alcohol, Chloroform,Glacial acetic acid
Carnoy’s fixative
Fixes Nissl granules and Cytoplasmic granules
Carnoy’s fixative
Recommended for mucopolysaccharide and nuclear proteins
Newcomer’s
Acts as both nuclear and biochemical fixative
Newcomer’s
Osmium Tetroxide is AKA
Osmic Acid
Produce pale yellow powder that dissolves in water (6% at 20 Degree Celsius)
Osmium Tetroxide
Fixes and stains unconjugated fats which appear Black
Osmium Tetroxide
Secondary fixative for EM after aldehydes
Osmium Tetroxide
Preserves mitochondria and Golgi bodies
Osmium Tetroxide
Osmium Tetroxide, Regaud, Potassium Dichromate all fixes
Mitochondria
Regaud’s and Osmium tetroxide both fixes
Golgi bodies
It is expensive, cause Corneal blindness and inhibit hematoxylin
Osmium tetroxide
Osmium tetroxide ihibits
Hematoxylin
Fixative that contains Osmium tetroxide
Flemming’s
Most common chrome-osmium acid fixative
Flemming’s
Excellent fixative for nuclear strctures
Flemming’s or Flemming’s w/o Acetic acid?
Flemming’s
For cytoplasmic structures especially mitochondria
Flemming’s or Flemming’s w/o Acetic acid?
Flemming’s w/o Acetic acid
Weak Decalcifying agent
TCA
Used at Ice cold temperature from -5 to 4 Degree Celsius
Acetone
Acetone is used at Ice cold temperature from
-5 to 4 Degree Celsius
For diffusible enzymes such as Phosphatases and Lipases
Acetone
For fixing brain tissues:Rabies
Acetone
Thermal coagulation of tissues proteins
Heat Fixation
For frozen tissue section and preparation of bacteriologic smears
Heat Fixation
Physical agent similar to heat(Oven)
Microwave technique
Increases movement of molecules and accelerate fixation,staining,decalcification,EM and immunohistochemistry
Microwave technique
Optimum temp for Microwave technique
45-55 Degree Celsius
Microwave technique can penetrate tissues with thickness of
10-15 mm
4% formalin ,Formol Saline, Acetone,Formalin for cryostat sections are all fixatives for
Enzymes Histochemistry
What are the fixatives for EM
Glutaraldehyde
Paraformaldehyde
Osmium tetroxide
Pallde’s Millonig’s, S.collidine are all contains
Osmium tetroxide
Optimum temp for EM fixation
4 Degree Celsius
Best Fixative for Electron Histochemistry and Electron Immunocytochemistry
Karnovsky’s
Karnovsky’s contains
Paraformaldehyde and Glutaraldehyde
Mixture of methanol and PEG;
Cost effective alternative to formalin;
Recover RNA,DNA, proteins for molecular analysis
UMFIX
Big size and Thicker
Retarded/ Enhaced?
Retarded
Presenced of Mucus
Retarded/ Enhaced?
Retarded
Presence of Pseudomucinous cyst of ovary
Retarded/ Enhaced?
Retarded
Remedy for the presence of Mucus
Wash with NSS
Presence of Fat
Retarded/ Enhaced?
Retarded
Presence of Blood
Retarded / Enhaced?
Retarded
Remedy for the presence of blood
Wash with NSS
Cold temperature
Retarded / Enhaced?
Retarded
Thinner and small size
Retarded / Enhaced?
Enhaced
Agitation
Retarded / Enhaced?
Enhaced
Agitation reduced processing time by how many percent?
30%
Heat
Retarded/ Enhanced?
Enhanced
Placing an already fixed tissue into another fixative to improve demonstration of substances and ensure further and complete hardening
Secondary Fixation
Secondary fixation that uses 2.5-3% Potassium Dichromate for 1 day
Post-Chromatization
Mordant used in Post-Chromatization for 1 day
2.5-3% Potassium Dichromate
Potassium Dichromate serves as what in post-chromatization
Mordant
Post-Chromatization is AKA
Post-Mordanting
Removal of excess fixative to improve staining and remove artifacts
Washing-out
Tap water can wash out?
Chromates, Formalin,Osmium tetroxide
50-70% alcohol can wash out
Picric Acid
Alcoholic iodine can wash out
Mercuric chloride
Transport medium for unfixed tissues (renal,skin,oral mucosa biopsies); refrigerated; not a fixative
Michel’s solution
Removal of calcium or lime salts from bones or calcified tissues following fixation
Decalcification
Decalcification has ________ concentrated acid solutions decalcify more rapidly but may destroy tissue
less or more
More
Ratio and Room of decalcification
20:1
Room temp
Heat and Agitation _____ decalcification
hastens/slows
hastens
Suspending the tissue in the decalcifying fluid hastens decalcification using what type of container
Metal / Glass
Glass due to acid agents
Duration of decalcification
1-2 days
Types of decalcifying agents
Acids
Chelating Agents
Ion Exchange Resins
Electrophoresis
“An ICE”
Widely used for routine decalcification
Acid Decalcifying agents
Types of Acid Decalcifying Agents
Nitric Acid
HCl
Formic Acid
TCA
Sulfurous Acid
Chromic Acid
Most common and rapid decalcifying agent
Nitric Acid
% of Nitric Acid
5-10%
Nitric Acid can be removed by
70% Alcohol
Remedy for nitric acid causing yellow color
Add 5% sodium thiosulfate / Urea crystals
This decalcifying agent imparts a yellow color due to formation of nitrous acid
Nitric Acid
Contains formalin and less tissue destruction;
Rapid for urgent biopsies
Formol Nitric Acid
Decalcifies and softens tissues at the same time
Perenyi’s Fluid
No maceration occurs because of chromic acid and alcohol
Perenyi’s fluid
